Developing multiplexed SNP assays with special reference to degraded DNA templates

This protocol describes a single nucleotide polymorphism (SNP) genotyping strategy for highly degraded DNA, using a two-stage multiplex whereby multiple fragments are first amplified in a single exponential reaction and the products of this PCR are added to a linear single-base-extension reaction. It utilizes the analytical power of a capillary electrophoresis system to simultaneously type all the target sites. The protocol is specifically written for use with severely fragmented templates, typical of ancient DNA, and can be adapted to widely used detection platforms. The addition of the single-phase genotyping step avoids the need for the re-amplification and cloning of PCR products, while providing its own controls for the detection of contamination and allelic drop-out. This protocol can facilitate the routine analysis of up to 52 SNP markers (haploid or diploid) in 96 samples in a single day, and is recommended for the authentication of data in all areas of DNA research (population and medical genetics, forensics, ancient DNA).     

A disposable and cost efficient microfluidic device for the rapid chip-based electrical detection of DNA

Requirements for a point-of-care device are an easy and robust read-out and – above all – a simple handling. We integrated an established robust electrical read-out for DNA-chips into a microfluidic device, thereby creating an automated analysis system that combines the necessary steps for a chip-based analysis. It is based on the electrical detection of biotin-labeled DNA in a gap between two microstructured electrodes on the surface of a DNA-chip. The biotin serves as binding molecule for streptavidin-conjugated horseradish peroxidase. A following enzyme-induced silver deposition bridges the gap by a conductive layer. The miniaturized chip gives the possibility to realize a durable system suitable for point-of-care applications.To enable an initial automation, all corresponding process steps were executed in a miniaturized silicone flow cell. The required defined temperatures for the hybridization and the washing steps can be adjusted by a heating foil.This paper characterizes the performance of the flow cell based system in terms of reaction speed and analysis time, sensitivity as well as specificity, and the comparison to a conventional system, without flow cell. These first steps of automation and integration will help to realize a laboratory-independent bioanalytical tool, for the use outside of specialized laboratories for fast analysis of different chemical and biological applications.     

Separation of phospholipids in microfluidic chip device: application to high-throughput screening assays for lipid-modifying enzymes

Phospholipid molecules such as ceramide and phosphoinositides play crucial roles in signal transduction pathways. Lipid-modifying enzymes including sphingomyelinase and phosphoinositide kinases regulate the generation and degradation of these lipid-signaling molecules and are important therapeutic targets in drug discovery. We now report a sensitive and convenient method to separate these lipids using microfluidic chip-based technology. The method takes advantage of the high-separation power of the microchips that separate lipids based on micellar electrokinetic capillary chromatography (MEKC) and the high sensitivity of fluorescence detection. We further exploited the method to develop a homogenous assay to monitor activities of lipid-modifying enzymes. The assay format consists of two steps: an on-plate enzymatic reaction using fluorescently labeled substrates followed by an on-chip MEKC separation of the reaction products from the substrates. The utility of the assay format for high-throughput screening (HTS) is demonstrated using phospholipase A(2) on the Caliper 250 HTS system: throughput of 80min per 384-well plate can be achieved with unattended running time of 5.4h. This enabling technology for assaying lipid-modifying enzymes is ideal for HTS because it avoids the use of radioactive substrates and complicated separation/washing steps and detects both substrate and product simultaneously.     

On the disruption of biochemical and biological assays by chemicals leaching from disposable laboratory plasticware

Plastic consumables, used universally in bioscience laboratories, are presumed inert with respect to bioassay outcomes. However, it is clear that many pipette tips, microfuge tubes, and other plastic disposables leach bioactive compounds into assay solutions, profoundly affecting data and experimental interpretation. In this paper we discuss the nature and sources of leachates and review several examples of compromised bioassay data that speak to the probable widespread nature of this largely unrecognised source of error. Strategies for minimizing leachate interferences are discussed.     

Application of a microfluidic device for counting of bacteria

AIMS: To develop a miniaturized analytical system for counting of bacteria. METHODS AND RESULTS: Escherichia coli cells were used throughout the experiments. The system consists of a microfluidic chamber, a fluorescence microscope with a charge-coupled device (CCD) camera and syringe pumps. The chamber was made of a silicone rubber (30 x 30 mm and 4 mm high). The E. coli cells were flowed from a micro-nozzle fabricated in the chamber and detected with the CCD camera. The individual cells were indicated as signal peaks on a computer. The cell counts showed a good correlation compared with that of a conventional plate counting method, and results of the simultaneous detection of live and dead cells were also presented. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The system having a small disposable nozzle has the advantages for low cost and safe medical or environmental analysis, when compared with a conventional flow cytometer. This is the first step of the development of a one-chip microbe analyzer.     

Cytophobic surface modification of microfluidic arrays for in situ parallel peptide synthesis and cell adhesion assays

A combination of PEG-based surface passivation techniques and spatially addressable SPPS (solid-phase peptide synthesis) was used to demonstrate a highly specific cell-peptide adhesion assay on a microfluidic platform. The surface of a silicon-glass microchip was modified to form a mixed self-assembled monolayer that presented PEG moieties interspersed with reactive amino terminals. The PEG provided biomolecular inertness and the reactive amino groups were used for consequent peptide synthesis. The cytophobicity of the surface was characterized by on-chip fluorescent binding assays and was found to be resistant to nonspecific attachment of cells and proteins. An integrated system for parallel peptide synthesis on this reactive amino surface was developed using photogenerated acid chemistry and digital microlithography. A constant synthesis efficiency of >98% was observed for up to 7mer peptides. To demonstrate specific cell adhesion on these synthetic peptide arrays, variations of a 7mer cell binding peptide that binds to murine B lymphoma cells were synthesized. Sequence-specific binding was observed on incubation with fluorescently labeled, intact murine B lymphoma cells, and key residues for binding were identified by deletional analysis.     

A solution to the problems of cytolysis assays with additional applications to other immunological and biochemical assays

After cellular immunoassays are compared with classical bioassays, conventional methods and consequent problems of data analysis for cytolysis assays are reviewed and a new solution is proposed. This solution incorporates new methods, called dose-response surface assays and analysis (DRSA), which estimate cytolytic activity coefficients on a surface in a three-dimensional space with two dose variables (killers and targets) and one response variable (counts). These new methods based on dose-response surfaces are demonstrated to be more informative and reliable than classical methods based on dose-response curves. In a test of the methods' robustness (sensitivity of parameter estimates to changes in the dose levels of the assay design), cytolytic activity coefficients estimated by DRSA varied by less than or equal to 30% over a reduction of three to four orders of magnitude in the dose levels. This remarkable robustness should be compared with the corresponding figures of as much as 500% over less than 1 order of magnitude for previously published results of coefficients estimated by conventional methods. DRSA is distinguished from replot-of-plots methods such as those used for enzyme inhibition assays in biochemistry, and is recommended as a more efficient method that should replace replot-of-plot methods now antiquated by the advent of microcomputers. DRSA can be applied to any experimental system that requires an activity coefficient to be estimated on a dose-response surface in a space of greater than or equal to 3 dimensions (greater than or equal to 2 dose variables and one response variable), regardless of the mathematical model and statistical estimators used to analyze the dose-response interaction. Finally, DRSA is compared with the methods known as response surface methodology (RSM), and is described as a new class of methods to be added to those that constitute RSM.     

Cell-type and donor-specific transcriptional responses to interferon-alpha. Use of customized gene arrays

A sensitive, specific, reproducible, robust, and cost-effective customized cDNA array system based on established nylon membrane technology has been developed for convenient multisample expression profiling for several hundred genes of choice. The genes represented are easily adjusted (depending on the availability of corresponding cDNAs) and the method is accordingly readily applicable to a wide variety of systems. Here we have focused on the expression profiles for interferon-alpha2a, the most widely used interferon for the treatment of viral hepatitis and malignancies, in primary cells (peripheral blood mononuclear cells, T cells, and dendritic cells) and cell lines (Kit255, HT1080, HepG2, and HuH7). Of 150 genes studied, only six were consistently induced in all cell types and donors, whereas 74 genes were induced in at least one cell type. IRF-7 was identified as the only gene exclusively induced in the hematopoietic cells. No gene was exclusively induced in the nonhematopoietic cell lines. In T cells 12, and in dendritic cells, 25 genes were induced in all donors whereas 45 and 42 genes, respectively, were induced in at least one donor. The data suggest that signaling through IFN-alpha2 can be substantially modulated to yield significant cell-type and donor-specific qualitative and quantitative differences in gene expression in response to this cytokine under highly standardized conditions.     

Novel LTCC-potentiometric microfluidic device for biparametric analysis of organic compounds carrying plastic antibodies as ionophores: application to sulfamethoxazole and trimethoprim

Monitoring organic environmental contaminants is of crucial importance to ensure public health. This requires simple, portable and robust devices to carry out on-site analysis. For this purpose, a low-temperature co-fired ceramics (LTCC) microfluidic potentiometric device (LTCC/μPOT) was developed for the first time for an organic compound: sulfamethoxazole (SMX). Sensory materials relied on newly designed plastic antibodies. Sol-gel, self-assembling monolayer and molecular-imprinting techniques were merged for this purpose. Silica beads were amine-modified and linked to SMX via glutaraldehyde modification. Condensation polymerization was conducted around SMX to fill the vacant spaces. SMX was removed after, leaving behind imprinted sites of complementary shape. The obtained particles were used as ionophores in plasticized PVC membranes. The most suitable membrane composition was selected in steady-state assays. Its suitability to flow analysis was verified in flow-injection studies with regular tubular electrodes. The LTCC/μPOT device integrated a bidimensional mixer, an embedded reference electrode based on Ag/AgCl and an Ag-based contact screen-printed under a micromachined cavity of 600 μm depth. The sensing membranes were deposited over this contact and acted as indicating electrodes. Under optimum conditions, the SMX sensor displayed slopes of about -58.7 mV/decade in a range from 12.7 to 250 μg/mL, providing a detection limit of 3.85 μg/mL and a sampling throughput of 36 samples/h with a reagent consumption of 3.3 mL per sample. The system was adjusted later to multiple analyte detection by including a second potentiometric cell on the LTCC/μPOT device. No additional reference electrode was required. This concept was applied to Trimethoprim (TMP), always administered concomitantly with sulphonamide drugs, and tested in fish-farming waters. The biparametric microanalyzer displayed Nernstian behaviour, with average slopes -54.7 (SMX) and +57.8 (TMP) mV/decade. To demonstrate the microanalyzer capabilities for real applications, it was successfully applied to single and simultaneous determination of SMX and TMP in aquaculture waters.     

Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45− cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2–400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.     

Simultaneous detection of antibacterial sulfonamides in a microfluidic device with amperometry

A highly sensitive and robust method for simultaneous detection of five sulfonamide drugs is developed by integrating the preconcentration and separation steps in a microfluidic device. An ampetrometry is performed for the selective detection of sulfonamides using an aluminum oxide-gold nanoparticle (Al(2)O(3)-AuNPs) modified carbon paste (CP) electrode at the end of separation channel. The preconcentration capacity of the channel is enhanced by using the field amplified sample stacking and the field amplified sample injection techniques. The experimental parameters affecting the analytical performances, such as pH, % of Al(2)O(3), volume of AuNPs, buffer concentration, and water plug length are optimized. A reproducible response is observed during the multiple injections of samples with RSDs<4%. The calibration plots are linear with the correlation coefficient between 0.991 and 0.997 over the range between 0.01 and 2025pM. The detection limits of five drugs are determined to be between 0.91 (±0.03) and 2.21 (±0.09)fM. The interference effects of common biological compounds are also investigated and the applicability of the method to the direct analysis of sulfonamides in real meat samples is successfully demonstrated. Long term stability of the modified electrode was also investigated.     

Measurements of elastic moduli of silicone gel substrates with a microfluidic device

Thin layers of gels with mechanical properties mimicking animal tissues are widely used to study the rigidity sensing of adherent animal cells and to measure forces applied by cells to their substrate with traction force microscopy. The gels are usually based on polyacrylamide and their elastic modulus is measured with an atomic force microscope (AFM). Here we present a simple microfluidic device that generates high shear stresses in a laminar flow above a gel-coated substrate and apply the device to gels with elastic moduli in a range from 0.4 to 300 kPa that are all prepared by mixing two components of a transparent commercial silicone Sylgard 184. The elastic modulus is measured by tracking beads on the gel surface under a wide-field fluorescence microscope without any other specialized equipment. The measurements have small and simple to estimate errors and their results are confirmed by conventional tensile tests. A master curve is obtained relating the mixing ratios of the two components of Sylgard 184 with the resulting elastic moduli of the gels. The rigidity of the silicone gels is less susceptible to effects from drying, swelling, and aging than polyacrylamide gels and can be easily coated with fluorescent tracer particles and with molecules promoting cellular adhesion. This work can lead to broader use of silicone gels in the cell biology laboratory and to improved repeatability and accuracy of cell traction force microscopy and rigidity sensing experiments.     

Aragonite crystallization in a Christmas-tree model microfluidic device with the addition of magnesium ions

Aragonite is an important dimorph of calcium carbonate, industrially and biologically. However, aragonite is so thermodynamically unstable that it is difficult to understand its formation mechanism. A continuous microfluidic system was employed, in which crystallization was induced only by diffusion in a micron-scale channel. Calcium carbonate (CaCO₃) formed by liquid-liquid reaction and magnesium ions (Mg²⁺) were used as additives. To assess the influence of Mg²⁺ concentration, the Mg²⁺/Ca²⁺ molar ratio was set to 1, 3, and 5. Laminar streams flowed in the detection channel with different concentration gradients. The initial crystallization time (t_I.C) increased exponentially and the density of crystals decreased as the Mg²⁺ ion concentration increased. Following transformation of all particles into snowman or sphere shapes, they became spinose sphere-shaped crystals, which was the final form in this study.     

Application of biochemical and polymerase chain reaction assays for identification of Campylobacter isolates from non-human primates

A multiplex polymerase chain reaction (PCR) assay was performed on 167 thermophilic campylobacters isolated from non-human primates. Samples were first identified by phenotypic methods resulting in 64 Campylobacter jejuni and 103 C. coli strains. Four strains identified biochemically as C. coli, were then determined to be C. jejuni by PCR. Comparison of methodologies showed that the main discrepancies were attributed to the hippurate hydrolysis test and sensitivity to cephalothin and nalidixic acid. Analysis of data showed that the application of phenotypic methods should be supplemented by a molecular method to offer a more reliable Campylobacter identification.     

Geometrical focusing of cells in a microfluidic device: an approach to separate blood plasma

It is well known that when a suspension of cells flows in small vessels (arterioles or venules), there exists a cell-free layer of a few microns adjacent to the vascular walls. Using an in vitro model, we show experimentally that for a fixed flow rate a geometrical constriction in the flow can artificially enhance the cell-free layer. Also, we show that rapid variation of the geometry coupled to the deformability of the cells can dramatically modify their spatial distribution in the channel. The effects of the constriction geometry, flow rate, suspending fluid viscosity, cell concentration, and cell deformability are studied and the results are interpreted in terms of a model of the hydrodynamic drift of an ellipsoidal cell in a shear flow. We propose a microfluidic application of this focusing effect for separation of the red blood cells from the suspending plasma.     

Separation and simultaneous detection of anticancer drugs in a microfluidic device with an amperometric biosensor

A simple and highly sensitive method for simultaneous detection of anticancer drugs is developed by integrating the preconcentration and separation steps in a microfluidic device with an amperometric biosensor. An amperometric detection with dsDNA and cardiolipin modified screen printed electrodes are used for the detection of anticancer drugs at the end of separation channel. The preconcentration capacity is enhanced thoroughly using field amplified sample stacking and field amplified sample injection techniques. The experimental parameters affecting the analytical performances, such as pH, temperature, buffer concentration, water plug length, and detection potential are optimized. A reproducible response is observed during multiple injections of samples with a RSD <5%. The calibration plots are linear with the correlation coefficient between 0.9913 and 0.9982 over the range of 2-60 pM. The detection limits of four drugs are determined to be between 1.2 (± 0.05) and 5.5 (± 0.3) fM. The applicability of the device to the direct analysis of anticancer drugs is successfully demonstrated in a real spiked urine sample. Device was also examined for interference effect of common chemicals present in real samples.     

Application of the MultiScreen system to cytokine radioreceptor assays

Radioreceptor assays are becoming increasingly valuable in the biotechnology community for a variety of basic and applied research applications. It is clear, for example, that assessing the potential spectrum of biological activities of a novel polypeptide regulatory factor can be greatly simplified by the development of a rapid radioreceptor assay, since a wide variety of cell types can be screened using a single type of assay. By contrast, searching for potentially diverse biological effects can be an extremely time-consuming process. In addition, screening for agonists/antagonists for hormones using radioreceptor assays has a marked advantage compared with biological assays, in that compounds or natural products that are toxic to cells will not read out as false positives in a binding assay. Our laboratory has developed a major program centered on the molecular characterization of receptors for polypeptide hormones involved in immune regulation, including a number of cytokines/interleukins and also several colony stimulating factors. We have developed a variety of radioreceptor- and fluorescence-based assay systems for ligand-receptor interactions, with applications in basic characterization, purification, cDNA cloning, and drug development screens for cytokine receptors. In this report we compare two assay formats, a standard phthalate oil centrifugation method and a novel plate filtration system, using the interaction between interleukin-1 alpha and its receptor as a test system.